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The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern 1 . For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings 2 . Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the m 2 6 A 2058 nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance. Structure-guided design and component-based synthesis are used to produce iboxamycin, a novel ribosome-binding antibiotic with potent activity against Gram-positive and Gram-negative bacteria.

The lincoamide antibiotic lincomycin, derived from Streptomyces lincolnensis , is widely used for the treatment of infections caused by gram-positive bacteria. As a common global regulatory factor of GntR family, DasR usually exists as a regulatory factor that negatively regulates antibiotic synthesis in Streptomyces . However, the regulatory effect of DasR on lincomycin biosynthesis in S. lincolnensis has not been thoroughly investigated. The present study demonstrates that DasR functions as a positive regulator of lincomycin biosynthesis in S. lincolnensis , and its overexpression strain OdasR exhibits a remarkable 7.97-fold increase in lincomycin production compared to the wild-type strain. The effects of DasR overexpression could be attenuated by the addition of GlcNAc in the medium in S. lincolnensis . Combined with transcriptome sequencing and RT-qPCR results, it was found that most structural genes in GlcNAc metabolism and central carbon metabolism were up-regulated, but the lincomycin biosynthetic gene cluster ( lmb ) were down-regulated after dasR knock-out. However, DasR binding were detected with the DasR responsive elements ( dre ) of genes involved in GlcNAc metabolism pathway through electrophoretic mobility shift assay, while they were not observed in the lmb . These findings will provide novel insights for the genetic manipulation of S. lincolnensis to enhance lincomycin production. Key points • DasR is a positive regulator that promotes lincomycin synthesis and does not affect spore production • DasR promotes lincomycin production through indirect regulation • DasR correlates with nutrient perception in S. lincolnensis

The adverse effects of short-term megadose of antibiotics exposure on the gastrointestinal and liver tissue reactions in young children have been reported. Antibiotic-induced intestinal and liver reactions are usually unpredictable and present a poorly understood pathogenesis. It is, therefore, necessary to develop strategies for reducing the adverse effects of antibiotics. Studies on the harm and rescue measures of antibiotics from the perspective of the gut–liver system are lacking. Here, we demonstrate that lincomycin exposure reduced body weight, disrupted the composition of gut microbiota and intestinal morphology, triggered immune-mediated injury and inflammation, caused liver dysfunction, and affected lipid metabolism. However, baicalin administration attenuated the lincomycin-induced changes. Transcriptome analysis showed that baicalin improved immunity in mice, as evidenced by the decreased levels of intestinal inflammatory cytokines and expression of genes that regulate Th1, Th2, and Th17 cell differentiation, and inhibited mucin type O-glycan biosynthesis pathways. In addition, baicalin improved liver function by upregulating the expression of genes involved in bile acid secretion and lipid degradation, and downregulating genes involved in lipid synthesis in lincomycin-treated mice. Bile acids can regulate intestinal immunity and strengthen hepatoenteric circulation. In addition, baicalin also improved anti-inflammatory bacteria abundance (Blautia and Coprobacillus) and reduced pathogenic bacteria abundance (Proteobacteria, Klebsiella, and Citrobacter) in lincomycin-treated mice. Thus, baicalin can ameliorate antibiotic-induced injury and its associated complications such as liver disease.

Lincomycin belongs to the antibiotics commonly used in veterinary medicine. Its residues are easily spread in the environment because of its physicochemical properties, including resistance to biodegradation and good solubility in water. One of the effective methods for the removal of lincomycin from wastewater is the photocatalytic process, but it is not widely used due to the price of photocatalysts. The aim of this work was to compare the photocatalytic efficiency and the mechanism of lincomycin degradation initiated by UVa radiation in the presence of TiO2-P25 and ZnO, as well as in the presence of industrial pigments commonly used in construction and containing TiO2. Lincomycin was found to undergo efficient photocatalytic degradation in the presence of a commercial TiO2-P25 photocatalyst, industrial pigments containing only anatase, and in the presence of ZnO. On the contrary, industrial pigments containing only rutile or a mixture of rutile and anatase practically did not show any photocatalytic activity. The composition of the solutions after the degradation of lincomycin in the presence of TiO2-P25 and ZnO differed significantly. Most of the identified organic degradation products contained conserved pharmacophores, and some of them could have been highly ecotoxic.

Mycoplasma synoviae (MS) is a highly prevalent bacterial species in poultry causing disease and severe economic losses. Antibiotic treatment is one of the control strategies that can be applied to contain clinical outbreaks in MS-free flocks, especially because this bacterium can be transmitted in ovo. It becomes, then, very important for veterinarians to know the antibiotic susceptibility of the circulating strains in order to choose the most appropriate first-line antibiotic molecule as a proactive role in fighting antibiotic resistance. We evaluated the Minimum Inhibitory Concentrations (MICs) of enrofloxacin, oxytetracycline, doxycycline, erythromycin, tylosin, tilmicosin, spiramycin, tiamulin, florfenicol and lincomycin for MS isolates collected between 2012 and 2017 in Italy. A total of 154 MS isolates from different poultry commercial categories (broiler, layer, and turkey sectors) was tested using commercial MIC plates. All MS isolates showed very high MIC values of erythromycin (MIC90 ≥8 μg/mL) and enrofloxacin (MIC90 ≥16 μg/mL). MIC values of doxycycline and oxytetracycline obtained were superimposable to each other with only a one-fold dilution difference. Discrepancies between MIC values of tylosin and tilmicosin were observed. Interestingly, seven isolates showed very high MIC values of lincomycin and tilmicosin, but not all of them showed very high MIC values of tylosin. Most of the MS isolates showed low MIC values of spiramycin, but seven strains showed a MIC ≥16 μg/mL. In the observation period, the frequency of the different MIC classes varied dependently on the tested antibiotic. Interestingly, tilmicosin MICs clearly showed a time-dependent progressive shift towards high-concentration classes, indicative of an on-going selection process among MS isolates. Until standardized breakpoints become available to facilitate data interpretation, it will be fundamental to continue studying MIC value fluctuations in the meantime in order to create a significant database that would facilitate veterinarians in selecting the proper drug for treating this impactful Mycoplasma.

Resistance proteins are perceived as mechanisms protecting bacteria from the inhibitory effect of their produced antibiotics or antibiotics from competitors. Here, we report that antibiotic resistance proteins regulate lincomycin biosynthesis in response to subinhibitory concentrations of antibiotics. In natural environments, antibiotics are important means of interspecies competition. At subinhibitory concentrations, they act as cues or signals inducing antibiotic production; however, our knowledge of well-documented antibiotic-based sensing systems is limited. Here, for the soil actinobacterium Streptomyces lincolnensis , we describe a fundamentally new ribosome-mediated signaling cascade that accelerates the onset of lincomycin production in response to an external ribosome-targeting antibiotic to synchronize antibiotic production within the population. The entire cascade is encoded in the lincomycin biosynthetic gene cluster (BGC) and consists of three lincomycin resistance proteins in addition to the transcriptional regulator LmbU: a lincomycin transporter (LmrA), a 23S rRNA methyltransferase (LmrB), both of which confer high resistance, and an ATP-binding cassette family F (ABCF) ATPase, LmrC, which confers only moderate resistance but is essential for antibiotic-induced signal transduction. Specifically, antibiotic sensing occurs via ribosome-mediated attenuation, which activates LmrC production in response to lincosamide, streptogramin A, or pleuromutilin antibiotics. Then, ATPase activity of the ribosome-associated LmrC triggers the transcription of lmbU and consequently the expression of lincomycin BGC. Finally, the production of LmrC is downregulated by LmrA and LmrB, which reduces the amount of ribosome-bound antibiotic and thus fine-tunes the cascade. We propose that analogous ABCF-mediated signaling systems are relatively common because many ribosome-targeting antibiotic BGCs encode an ABCF protein accompanied by additional resistance protein(s) and transcriptional regulators. Moreover, we revealed that three of the eight coproduced ABCF proteins of S. lincolnensis are clindamycin responsive, suggesting that the ABCF-mediated antibiotic signaling may be a widely utilized tool for chemical communication. IMPORTANCE Resistance proteins are perceived as mechanisms protecting bacteria from the inhibitory effect of their produced antibiotics or antibiotics from competitors. Here, we report that antibiotic resistance proteins regulate lincomycin biosynthesis in response to subinhibitory concentrations of antibiotics. In particular, we show the dual character of the ABCF ATPase LmrC, which confers antibiotic resistance and simultaneously transduces a signal from ribosome-bound antibiotics to gene expression, where the 5′ untranslated sequence upstream of its encoding gene functions as a primary antibiotic sensor. ABCF-mediated antibiotic signaling can in principle function not only in the induction of antibiotic biosynthesis but also in selective gene expression in response to any small molecules targeting the 50S ribosomal subunit, including clinically important antibiotics, to mediate intercellular antibiotic signaling and stress response induction. Moreover, the resistance-regulatory function of LmrC presented here for the first time unifies functionally inconsistent ABCF family members involving antibiotic resistance proteins and translational regulators.

Plants and algae adapt to fluctuating light conditions to optimize photosynthesis, minimize photodamage, and prioritize energy investments. Changes in the translation of chloroplast mRNAs are known to contribute to these adaptations, but the scope and magnitude of these responses are unclear. To clarify the phenomenology, we used ribosome profiling to analyze chloroplast translation in maize seedlings following dark-to-light and light-to-dark shifts. The results resolved several layers of regulation. (i) The psbA mRNA exhibits a dramatic gain of ribosomes within minutes after shifting plants to the light and reverts to low ribosome occupancy within one hour in the dark, correlating with the need to replace damaged PsbA in Photosystem II. (ii) Ribosome occupancy on all other chloroplast mRNAs remains similar to that at midday even after 12 hours in the dark. (iii) Analysis of ribosome dynamics in the presence of lincomycin revealed a global decrease in the translation elongation rate shortly after shifting plants to the dark. The pausing of chloroplast ribosomes at specific sites changed very little during these light-shift regimes. A similar but less comprehensive analysis in Arabidopsis gave similar results excepting a trend toward reduced ribosome occupancy at the end of the night. Our results show that all chloroplast mRNAs except psbA maintain similar ribosome occupancy following short-term light shifts, but are nonetheless translated at higher rates in the light due to a plastome-wide increase in elongation rate. A light-induced recruitment of ribosomes to psbA mRNA is superimposed on this global response, producing a rapid and massive increase in PsbA synthesis. These findings highlight the unique translational response of psbA in mature chloroplasts, clarify which steps in psbA translation are light-regulated in the context of Photosystem II repair, and provide a foundation on which to explore mechanisms underlying the psbA-specific and global effects of light on chloroplast translation.

Disruption of protein homeostasis in chloroplasts impairs the correct functioning of essential metabolic pathways, including the methylerythritol 4-phosphate (MEP) pathway for the production of plastidial isoprenoids involved in photosynthesis and growth. We previously found that misfolded and aggregated forms of the first enzyme of the MEP pathway are degraded by the Clp protease with the involvement of Hsp70 and Hsp100/ClpC1 chaperones in Arabidopsis thaliana. By contrast, the combined unfolding and disaggregating actions of Hsp70 and Hsp100/ClpB3 chaperones allow solubilization and hence reactivation of the enzyme. The repair pathway is promoted when the levels of ClpB3 proteins increase upon reduction of Clp protease activity in mutants or wild-type plants treated with the chloroplast protein synthesis inhibitor lincomycin (LIN). Here we show that LIN treatment rapidly increases the levels of aggregated proteins in the chloroplast, unleashing a specific retrograde signaling pathway that up-regulates expression of ClpB3 and other nuclear genes encoding plastidial chaperones. As a consequence, folding capacity is increased to restore protein homeostasis. This sort of chloroplast unfolded protein response (cpUPR) mechanism appears to be mediated by the heat shock transcription factor HsfA2. Expression of HsfA2 and cpUPR-related target genes is independent of GUN1, a central integrator of retrograde signaling pathways. However, double mutants defective in both GUN1 and plastome gene expression (or Clp protease activity) are seedling lethal, confirming that the GUN1 protein is essential for protein homeostasis in chloroplasts.

Mycoplasma ( M .) hyosynoviae is a facultative pathogen, causing arthritis in finisher pigs world-wide. In the absence of a commercial vaccine improvement of housing conditions and antibiotic therapy are the only options to alleviate the clinical signs. This study aimed to determine antibiotic susceptibility profiles of 106 M . hyosynoviae isolates against ten antibiotics licensed for veterinary use in cases of arthritis. The isolates were collected between 2018 and 2023 from five European countries: Austria (n = 20), Belgium (n = 20), Germany (n = 25), Hungary (n = 21) and Italy (n = 20). The minimal inhibitory concentrations (MIC) were determined by broth micro-dilution assay. The tested isolates were highly susceptible to tiamulin (MIC 90  ≤ 0.039 µg/ml), tylvalosin (MIC 90  ≤ 0.039 µg/ml) and lincomycin (MIC 90  ≤ 0.25 µg/ml). Low concentrations of tylosin (MIC 90 0.5 µg/ml) and tilmicosin (MIC 90 1 µg/ml) inhibited the growth of the isolates. While moderate minimal inhibitory concentrations were detected for doxycycline (MIC 90 0.312 µg/ml), oxytetracycline (MIC 90 2 µg/ml), enrofloxacin (MIC 90 0.625 µg/ml) and florfenicol (MIC 90 2 µg/ml), only high concentrations of tulathromycin (MIC 90 64 µg/ml) inhibited the growth of the isolates. Statistical analysis revealed significant differences between countries in case of enrofloxacin, where the Hungarian isolates showed the lowest MIC values, and the German isolates the highest MIC values among the tested countries. Our results show that European M. hyosynoviae isolates are generally susceptible to the tested antibiotics with the exception of tulathromycin. The country specific differences indicate the importance of regular susceptibility testing of isolates on a Pan-European level.

The spread of antibiotic resistance through horizontal gene transfer (HGT) in food-associated bacteria represents an emerging public health concern. Staphylococcus equorum strain KS1030, isolated from a high-salt fermented food, carries plasmids encoding the lincomycin resistance gene lnuA and the relaxase gene rlx , both of which contribute to resistance dissemination. Previous studies have shown that strain KS1030 can transfer the lnuA gene both within and across subspecies when exposed to lincomycin. To investigate the transcriptional basis of this phenomenon, we performed RNA sequencing (RNA-Seq) to analyze the global gene expression profile of KS1030 under lincomycin stress (30 mg/L). Transcriptome analysis revealed more differentially expressed genes (DEGs) at 2 h than at 4 h, with enriched categories including amino acid transport and metabolism (22.9%), transcription (19.3%), and inorganic ion transport and metabolism (14.7%). Genes involved in ornithine, Fe³⁺, siderophore, and tryptophan metabolism, as well as stress regulators such as sigB , dcuSR , and helix-turn-helix transcriptional regulators, were strongly induced. Genome analysis further identified the competence (Com) operon and DNA translocase ( ftsK ) as potential transport systems, with comGC classified as a DEG. To capture short-term dynamics not resolved by RNA-Seq, quantitative real-time PCR was performed at 30-min intervals. Several genes, including comC , comEC , comFA , and ftsK , peaked at 1.5 h, while lnuA and rlx peaked at 1 h. Although the roles of the Com and FtsK systems in HGT remain unresolved, their induction under lincomycin stress suggests a potential contribution to plasmid transfer, offering new insight into the adaptive and gene transfer responses of S. equorum. However, as this study relies solely on transcriptional data from a single strain and antibiotic condition, functional validation—such as targeted gene disruption—will be required to confirm the involvement of these candidate HGT-related genes.